Boronic acid or borinic acid derivatives as enzyme stabilizers

ABSTRACT

The invention relates to a liquid composition which comprises an enzyme and a enzyme stabilizing component. The enzyme stabilizing component is a boronic acid derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional application of U.S. application Ser. No.08/571,954 filed Jan. 25, 1996 now U.S. Pat. No. 5,840,677, which is acontinuation of PCT/DK94/00249 filed Jun. 22, 1994 and claims priorityunder 35 U.S.C. 119 of Danish applications 833/93 and 477/94 filed Jul.9, 1993 and Apr. 26, 1994, respectively, the contents of which are fullyincorporated herein by reference.

FIELD OF INVENTION

This invention relates to a liquid composition containing an enzyme andan improved enzyme stabilizer.

BACKGROUND OF THE INVENTION

Storage stability problems are well known with liquids containingenzyme(s). Especially in enzyme-containing liquid detergents a majorproblem, in particular if the detergent contains protease, is that ofensuring enzyme activity over time.

The prior art has dealt extensively with improving the storagestability, for example by adding a protease inhibitor.

Boric acid and boronic acids are known to reversibly inhibit proteolyticenzymes. A discussion of the inhibition of one serine protease,subtilisin, by boronic acid is provided in Molecular & CellularBiochemistry 51, 1983, pp. 5-32.

Boronic acids have very different capacities as subtilisin inhibitors.Boronic acids containing only alkyl groups such as methyl, butyl or2-cyclohexylethyl are poor inhibitors with methylboronic acid as thepoorest inhibitor, whereas boronic acids bearing aromatic groups such asphenyl, 4-methoxyphenyl or 3,5-dichlorophenyl are very good inhibitorswith 3,5-dichlorophenylboronic acid as a particularly effective one (seeKeller et al, Biochem. Biocphys. Res. Com. 176, 1991, pp. 401-405).

It is also claimed that aryl boronic acids which have a substitution atthe 3-position relative to boron are unexpectedly good reversibleprotease inhibitors. Especially, acetamidobenzene boronic acid isclaimed to be a superior inhibitor of proteolytic enzymes (see WO92/19707).

The inhibition constant (K_(i)) is ordinarily used as a measure ofcapacity to inhibit enzyme activity, with a low K_(i) indicating a morepotent inhibitor. However, it has earlier been found that the K_(i)values of boronic acids do not always tell how effective inhibitors theyare (see for instance WO 92/19707).

SUMMARY OF THE INVENTION

In this invention it is surprisingly found that some special boronic andborinic acid derivatives have extraordinary good capacities as enzymestabilizers.

Accordingly, the present invention relates to a liquid compositioncomprising an enzyme and a boronic acid or a borinic acid derivativeenzyme stabilizer of the following formula: ##STR1## where R₁ is anoptionally substituted fused aromatic ring structure containing 14 or 18carbon atoms in the ring, or an optionally substituted monocyclic orfused aromatic hetero-cyclic ring structure containing up to 17 carbonatoms in the ring, or an optionally substituted monocyclic or fusedquinon-oid ring structure containing up to 18 carbon atoms in the ring;R₂ has the formula: ##STR2## where X is the same or different andselected from hydrogen, C₁ -C₆ alkyl, substituted C₁ -C₆ alkyl, aryl,substituted aryl, hydroxy, hydroxyl derivative, halogen, amine,alkylated amine, amine derivative, nitro, thiol, thiol derivative,aldehyde, acid, acid salt, ester, sulfonate or phosphonate, and o,p andq may be the same or different and may each be 0,1 or 2; m and n may bethe same or different and may each be 0 or 1; R₃ is the same ordifferent as R₁ and selected from R₁, or R₃ is a hydroxyl group, or R₁and R₃ are both optionally substituted monocyclic or dicyclic aromaticring structures.

In this context optionally substituted ring structures mean that thesubstituents on the ring structure are freely chosen, but they arepreferably selected from hydrogen, C₁ -C₆ alkyl, substituted C₁ -C₆alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen,amine, alkylated amine, amine derivative, nitro, thiol, thiolderivative, aldehyde, acid, acid salt, ester, sulfonate or phosphonate.

DETAILED DISCLOSURE OF THE INVENTION Preparation of Boronic and BorinicAcid Derivatives

Boronic and borinic acid derivatives may be prepared using methods wellknown to those skilled in the art, for example by using one of thefollowing methods:

a) Hydroboration of unsaturated materials, i.e. alkenes and alkynes,using either catecholborane (1,3,2-benzodioxaborole) ordichloroborane-dimethyl-sulphide complex as the hydroborating agent, forreference see H. C. Brown, S. K. Gupta in JACS 97, 1975, pp. 5249-5255and H. C. Brown, N. Ravindran, S. U. Kuikarni in J.Orq.Chem. 45, (1980),p. 384.

b) The reaction of a Grignard reagent with either tri-n-butylborate ortrimethylborate, followed by hydrolysis of the boronic ester thusformed, for reference see F. R. Bean, J. R. Johnson in Jacs 54, 1932,pp. 4415-4425 and S. H. Dandegaonher, S. P. Ingleshwar in Journal ofShivasi University 6, 1932, pp. 11-13. Bromo-starting materials that arenot commercially available may be prepared conveniently in two stepsfrom the corresponding carboxylic acids by reduction with LiAlH₄,followed by treatment with CBr₄.

c) The reaction of an organolithium reagent with butylborate, forreference see S. O. Lauesson, pp. 387-395 in Thionpene Chemistry, part 7and D. Florentin, B. Roques in C.R.Acad.Sc.Paris, t.270 (May 11, 1970),pp. 1608-1610.

d) Borinic acid derivatives are prepared according to method b. However,the ratio of Grignard reagent to borate adopted is 2:1.

e) Any nuclear substitution or protection of functional groups isachieved by using standard methods well known to those skilled in theart.

Stabilizers

According to the invention the liquid composition may contain up to 500mM of the stabilizer (the boronic or the borinic acid derivative),preferably the liquid composition may contain 0.001-250 mM of thestabilizer, more preferably the liquid composition may contain 0.005-100mM of the stabilizer, most preferably the liquid composition may contain0.01-10 mM of the stabilizer.

Enzymes

According to the invention the liquid composition contains at least oneenzyme. The enzyme may be any commercially available enzyme, inparticular an enzyme selected from the group consisting of proteases,amylases, lipases, cellulases or peroxidases or any mixture thereof.Mixtures of enzymes from the same class (e.g. lipases) are alsoincluded.

The amount of enzyme used in the composition varies according to thetype of enzyme(s) and the use intended. If the liquid is a detergentliquid, the amount of each enzyme will typically be 0.2-40 μM,especially 0.4-20 μM (generally 5-1000 mg/l, especially 10-500 mg/l)calculated as pure enzyme protein.

Protease: Any protease suitable for use in a liquid composition can beused. Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically or geneticallymodified mutants are included. It may be a serine protease, preferablyan alkaline microbial protease or a trypsin-like protease. Examples ofalkaline proteases are subtilisins, especially those derived fromBacillus, e.g. subtilisin Novo, subtilisin Carlsberg, subtilisin 309,subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examplesof commercial Bacillus subtilisins are Alcalase®, Savinase®, Esperase®and Durazym™ products of Novo Nordisk A/S. Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270.

Amvlase: Any amylase suitable for use in a liquid composition can beused. Suitable amylases include those of bacterial and fungal origin.Chemically or genetically modified mutants are included. Amylasesinclude, for example, α-amylases obtained from a special strain of B.licheniformis, described in more detail in British Patent SpecificationNo. 1,296,839. Particularly preferred is Termamyl®, available from NovoNordisk A/S.

Lioase: Any lipase suitable for use in a liquid composition can be used.Suitable lipases include those of bacterial and fungal origin.Chemically or genetically modified mutants are included. Particularlypreferred is lipase obtained by cloning the gene from Humicolalanuginosa and expressing the gene in Aspercillus orvzae, as describedin EP 0 258 068, available under the trade mark Lipolase® from NovoNordisk A/S.

Cellulase: Any cellulase suitable for use in a liquid composition can beused. Suitable cellulases include those of bacterial and fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307. Particularlypreferred is Celluzyme™ produced by a strain of Humicola insolens,available from Novo Nordisk A/S.

Peroxidase: Any peroxidase suitable for use in a liquid detergentcomposition can be used herein. Suitable peroxidases herein includethose of plant, bacterial and fungal origin. Chemically or geneticallymodified mutants are included. Examples of suitable peroxidases arethose derived from a strain of Coprinus, e.g. C. cinerius or C.macrorhizus, or from a strain of Bacillus, e.g. B. pumilus, particularlyperoxidase according to PCT/DK90/00260.

Detergents

If the liquid composition of the invention is a detergent, it willbeside enzyme(s) and stabilizer comprise a surfactant.

The liquid detergent may be aqueous, typically containing up to 70% ofwater and 0-30% of organic solvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂ O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of e.g. the amide, imide, orsulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative as e.g. an aromatic borate ester,and the composition may be. formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners, orperfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. 7-11.

Particular forms of detergent compositions within the scope of theinvention include:

1) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                       15-21%                                                   (calculated as acid)                                                          alcohol ethoxylate   12-18%                                                   (e.g. C.sub.12-15 alcohol, 7 EO or                                            C.sub.12-15 alcohol, 5 EO)                                                    soap as fatty acid (e.g. oleic acid)                                                                3-13%                                                   alkenylsuccinic acid (C.sub.12-14)                                                                  0-13%                                                   aminoethanol          8-18%                                                   citric acid          2-8%                                                     phosphonate          0-3%                                                     polymers (e.g. PVP, PEG)                                                                           0-3%                                                     borate (as B.sub.4 O.sub.7)                                                                        0-2%                                                     ethanol              0-3%                                                     propylene glycol      8-14%                                                   enzymes              0-5%                                                     minor ingredients    0-5%                                                     (e.g. dispersants, suds suppressors,                                          perfume, optical brightener)                                                  ______________________________________                                    

2) An aqueous structured liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                          15-21%                                                (calculated as acid)                                                          alcohol ethoxylate      3-9%                                                  (e.g. C.sub.12-15 alcohol, 7 EO                                               or C.sub.12-15 alcohol, 5 EO)                                                 soap as fatty acid (e.g. oleic acid)                                                                   3-10%                                                zeolite (as NaAlSiO.sub.4)                                                                            14-22%                                                potassium citrate        9-18%                                                borate (as B.sub.4 O.sub.7)                                                                           0-2%                                                  carboxymethylcellulose  0-2%                                                  polymers (e.g PEG, PVP) 0-3%                                                  anchoring polymers as   0-3%                                                  e.g. lauryl metharylate/acrylic acid copolymer;                               molar ratio 25:1; MW 3800                                                     glycerol                0-5%                                                  enzymes                 0-5%                                                  minor ingredients       0-5%                                                  (e.g. dispersants, suds suppressors, perfume,                                 optical brighteners)                                                          ______________________________________                                    

3) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                          15-23%                                                (calculated as acid)                                                          alcohol ethoxysulfate    8-15%                                                (e.g. C.sub.12-15 alcohol, 2-3 EO)                                            alcohol ethoxylate      3-9%                                                  (e.g. C.sub.12-15 alcohol, 7 EO                                               or C.sub.12-15 alcohol, 5 EO)                                                 soap as fatty acid (e.g. lauric acid)                                                                 0-3%                                                  aminoethanol            1-5%                                                  sodium citrate           5-10%                                                hydrotrope (e.g. sodium toluenesulfonate)                                                             2-6%                                                  borate (as B.sub.4 O.sub.7)                                                                           0-2%                                                  carboxymethylcellulose  0-1%                                                  ethanol                 1-3%                                                  propylene glycol        2-5%                                                  enzymes                 0-5%                                                  minor ingredients (e.g. polymers, dispersants,                                                        0-5%                                                  perfume, optical brighteners)                                                 ______________________________________                                    

4) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                          20-32%                                                (calculated as acid)                                                          alcohol ethoxylate       6-12%                                                (e.g. C.sub.12-15 alcohol, 7 EO                                               or C.sub.12-15 alcohol, 5 EO)                                                 aminoethanol            2-6%                                                  citric acid              8-14%                                                borate (as B.sub.4 O.sub.7)                                                                           1-3%                                                  polymer (e.g. maleic/acrylic acid copolymer,                                                          0-3%                                                  anchoring polymers as e.g.                                                    lauryl methacrylate/acrylic acid                                              copolymer and CMC)                                                            glycerol                3-8%                                                  enzymes                 0-5%                                                  minor ingredients (e.g. hydrotropes,                                                                  0-5%                                                  dispersants, perfume, optical brighteners)                                    ______________________________________                                    

5) Detergent formulations as described in 1)-4) where the content oflinear alkylbenzenesulfonate--or a part of it--is substituted by alkylsulfate (C₁₂ -C₁₈).

6) Detergent formulations as described in 1)-5) which contain astabilized or encapsulated peracid either as an additional component oras a substitute for already specified bleach systems.

7) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant as e.g. linear alkoxylatedprimary alcohol, a builder system (e.g. phosphate), enzyme and alkali.The detergent may also comprise anionic surfactant and/or a bleachsystem.

Tests of Stabilizers

According to the invention the effectiveness of each stabilizer may betested in one or more of the following three tests:

a) Storage Stability Test in Liquid Detergent: Enzyme(s) and stabilizerare added to a liquid detergent formulation and stored at well definedconditions. The enzyme activity of each enzyme is determined as afunction of time, e.g. after 0, 3, 7 and 14 days.

To calculate the inhibition efficiency from the storage stability date areaction mechanism is proposed. The following reactions give arelatively simple, but yet plausible, mechanism for a liquid detergentcontaining protease (P), lipase (L), and inhibitor (I):

I) Autodigestion of protease: P+P→D_(P) +P

II) Denaturation of protease: P→D_(P)

III) Inhibition of protease: P+I=PI

IV) Protease digestion of inhibited enzyme: P+PI→P+D_(P) +I

V) Denaturation of inhibited enzyme: PI→D_(P) +I

VI) Protease digestion of lipase: P+L→P+D_(L)

VII) Denaturation of lipase: L→D_(L)

where D_(P) and D_(L) are denatured (i.e. non-active) protease andlipase.

From these reactions three coupled differential equations are deriveddescribing the deactivation of P, L and PI. The reaction rate constantsare derived from storage stability data by the use of a parameterestimation method (Gauss-Newton with the Levenberg modification). Thestorage stability data give the concentration of (P+PI) and L as afunction of time.

Reaction III is much faster than the other reactions and equilibrium isassumed in the calculations. Reaction IV is excluded from the system toreduce the number of parameters thereby describing the stability of theinhibited enzyme by only one reaction rate constant (from equation V).

In all experiments there is a large surplus of inhibitor moleculescompared to protease molecules, i.e. a constant concentration ofinhibitor (corresponding to the added amount of inhibitor) is areasonable assumption.

The specific values of the reaction rate constants are somewhatsensitive to small variations in the data, but the sensitivity isreduced significantly by giving the results relatively to the value fromBoric Acid. An improvement factor is thus derived: ##EQU1## IF_(I)measures the inhibition efficiency given by the inhibition constantsK_(I) from reaction III.

b) The "Milk" Test: In this test the stabilizer to be tested is comparedwith a reference inhibitor (boric acid). The test is described indetails below:

Preparation of "inhibitor" milk: 0.075 g of CaCl₂ (dried fine-granularpure, Merck), 0.16 g of 3,3-dimethylglutaric acid (SIGMA) and 2.5 mmoleof stabilizer/inhibitor are weighed out and dissolved in 50 ml ofdemineralised water. pH is adjusted to approx. 6.0 with NaOH. 6.0 g ofskimmed milk powder (dehydrated, DIFCO Lab.) are weighed out in a 100 mlbeaker, and the solution of salt+buffer+stabilizer/inhibitor is added.This mixture is stirred heavily for some minutes to be sure that alllumps, if any, are apart. Thereafter the mixture is stirred for 30minutes. pH is adjusted to 6.50 with NaOH. Skimmed milk from a bottlecan be used instead of powder. Use milk from the same bottle for allstabilizers/inhibitors in one run.

Preparation of the enzyme: Prepare a solution of approx. 30 KNPU/litreof Savinase® (available from Novo Nordisk A/S) in boric acid buffer (seebelow). The Savinase activity is determined relatively to an enzymestandard. A folder AF 220/1-GB describing the analytical method ofdetermining the Savinase activity is available upon request to NovoNordisk A/S, Denmark, which folder is hereby included by reference.

Example: 1.0 g of 16 KNPU/g liquid Savinase is weighed out and 50 ml ofboric acid buffer are added. The mixture is stirred for 15 minutes. 10ml of this solution are filled into a 100 ml beaker, and boric acidbuffer is added up to 100 ml. Thereafter the mixture is stirred for 15minutes. Boric acid buffer: 2.5 g of boric acid (Merck) are dissolved in500 ml of demineralised water. pH is adjusted to 9.0 with NaOH.

The curdling: 10.0 ml of stabilizer/inhibitor are added to a test tube.3 test tubes of each stabilizer/inhibitor are made and placed in a 30°C. water bath. The test tubes are left in the water bath for one hour.1.00 ml of Savinase solution is added to the test tube and thestop-watch is started. The tube is mixed for 10 seconds on the"vibrator" and thereafter placed in the water bath. When the curdlingstarts the stop-watch is stopped. The deviation between the curdlingtime for the three test tubes should not be more than approx. 10seconds. How and when the curdling starts must be learned in practiceand the same person should curdle all samples. The curdling time for thereference inhibitor (boric acid) should be around 3-4 minutes (if thecurdling time is longer, a stronger protease solution should be used).The curdling time is approx. linear proportional to 1/(proteaseactivity). The result can be reported as an improvement factor IFdefined by: (curdling time stabilizer)/(curdling time reference).

c) Determination of K_(i) : The inhibition constant K_(i) may bedetermined by using standard methods, for reference see Keller et al,Biochem. Biochys. Res. Com. 176, 1991, pp.401-405; J. Bieth inBaver-Symposium "Proteinase Inhibitors", pp. 463-469, Springer-verlag,1974 and Lone Kierstein Hansen in "Determination of Specific Activitiesof Selected Detergent Proteases using Protease Activity, MolecularWeights Kinetic Parameters and Inhibition Kinetics", PhD-report, NovoNordisk A/S and University of Copenhagen, 1991.

The invention is further illustrated in the following examples which arenot intended to be in any way limiting to the scope of the invention asclaimed.

EXAMPLE 1 Preparation of 3-Thiopheneboronic Acid

3-Bromothiophene (0.043 m) in sodium dried ether (100 ml) was cooled to-60° C. Butyllithium (30 ml of 1 M) was added rapidly. The mixture wasthen stirred for 3 minutes, thereafter tri-n-butyl borate (0.043 m) ortrimethylborate (0.043 m) in sodium dried ether (25 ml) was added. Themixture was stirred for 4 hours and allowed to warm to room temperature.Thereafter the reaction mixture was treated with hydrochloric acid (1 M)and the ether layer was separated. The aqueous layer was extracted withether (2 times 25 ml). The combined ether layers were extracted withsodium hydroxide (1 M). The alkaline solution was then acidified withhydrochloric acid (10%), thus precipitating the desired boronic acid.The boronic acid was isolated and then recrystallized from water/ethanoland allowed to dry in air. C₄ H₅ BO₂ S, mpt. 163-1640° C.

Preparation of Diphenylborinic Acid.

This was prepared using the above method. The Grignard reagent wasprepared from bromobenzene and Magnesium turnings. However, two moles ofGrignard reagent were used per one mole of tri-n-butylborate. Theborinic acid so formed was isolated by reaction with ethanolamine thusyielding the diphenylborinic acid, ethanolamine complex ((C₆ H₅)₂ BO·CH₂CH₂ NH₂), which is easier to handle. Mpt.192-194° C.

EXAMPLE 2 Determination of K_(i)

The inhibition constants K_(i), for the inhibition of Alcalase andSavinase respectively, were determined using standard methods under thefollowing conditions:

Substrate:Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitro-anilide=SAAPFpNA(Sigma S-7388). Buffer: 0.1 M Tris-HCl pH 8.6; 25° C.

Enzyme concentration in assay: Alcalase: 1×10⁻¹⁰ -3×10⁻¹⁰ M Savinase:1×10⁻¹⁰ -3×10⁻¹⁰ M

The initial rate of substrate hydrolysis was determined at ninesubstrate concentrations in the range of 0.01 to 2 mM using a Cobas Faraautomated spectrophotometer. The kinetic parameters V_(max) and K_(m)were determined using ENZFITTER (a non-linear regression data analysisprogram). k_(cat) was calculated from the equation V_(max) =k_(cat) X[E_(o) ]. The concentration of active enzyme [E_(o) ] was determined byactive site titration using tight-binding protein proteinase inhibitors.The inhibition constant K_(i) was calculated from plots of K_(m)/k_(cat) as a function of the concentration of inhibitor. The inhibitorswere assumed to be 100% pure and the molar concentrations weredetermined using weighing numbers and molecular weights.

The results of the inhibition constants K_(i) of the boronic and borinicacid derivative enzyme stabilizers tested are listed below.

                  TABLE 1                                                         ______________________________________                                        The Inhibition Constants for the Inhibition of Alcalase and                   Savinase by Different Boronic and Borinic Acid Derivatives                    Boric Acid are included for Comparison.                                                         K.sub.i  K.sub.i                                            Inhibitor         Alcalase Savinase                                           ______________________________________                                        Boric acid        30 mM    20 mM                                              Thiophene-3-      2 mM     2 mM                                               boronic acid                                                                  Thiophene-2-      2 mM     2 mM                                               boronic acid                                                                  4-Methylthiophene-                                                                              1.8 mM   3 mM                                               2-boronic acid                                                                5-Ethylthiophene- 0.7 mM   0.9 mM                                             2-boronic acid                                                                5-Methylthiophene-                                                                              2 mM     3 mM                                               2-boronic acid                                                                5-Bromothiophene- 0.4 mM   0.2 mM                                             2-boronic acid                                                                5-Chlorothiophene-                                                                              0.3 mM   0.2 mM                                             2-boronic acid                                                                Dibenzothiophene- 0.9 mM   1.5 mM                                             1-boronic acid                                                                Dibenzofuran-1-   1.1 mM   1.5 mM                                             boronic acid                                                                  Dibenzofuran-4-   0.9 mM   1.1 mM                                             boronic acid                                                                  Picoline-2-boronic                                                                              3 mM     4 mM                                               acid                                                                          Diphenylborinic   2 mM     4 mM                                               acid (ethanolamine                                                            complex)                                                                      5-Methoxythiophene-2-                                                                           2 mM     1 mM                                               boronic acid                                                                  Thionaphthrene-1- 2 mM     3 mM                                               boronic acid                                                                  Furan-2-boronic   3 mM     4 mM                                               acid                                                                          Furan-3-boronic   10 mM    7 mM                                               acid                                                                          2,5-dimethyl-     3 mM     6 mM                                               thiophene-3-boronic                                                           acid                                                                          Benzofuran-1-boronic                                                                            1 mM     0.8 mM                                             acid                                                                          3-Methoxythiophene-2-                                                                           3 mM     1.2 mM                                             boronic acid                                                                  5-n-Propyl-       1.5 mM   3 mM                                               thiophene-2-                                                                  boronic acid                                                                  5-Methoxyfuran-2- 2 mM     3 mM                                               boronic acid                                                                  3-Bromothiophene- 2 mM     2 mM                                               2-boronic acid                                                                5-Ethylfuran-2-   2 mM     3 mM                                               boronic acid                                                                  4-Carbazole ethyl 1 mM     2 mM                                               boronic acid                                                                  ______________________________________                                    

EXAMPLE 3

Storage Stability Test in Liquid Detergent

Different boronic and borinic acid derivative enzyme stabilizers werealso tested in storage stability tests in liquid detergents using themethod described previously under the following conditions:

Detergent Base (US-type) % wt (as pure components)

    ______________________________________                                        Nansa 1169/p  10.3 (Linear Alkylbenzene Sulfonate, LAS)                       Berol 452      3.5 (Alkyl Ether Sulfate, AES)                                 Oleic acid     0.5                                                            Coconut fatty acid                                                                           0.5                                                            Dobanol 25-7   6.4 (Alcohol Ethoxylate, AEO)                                  Sodium xylene sulfonate                                                                      5.1                                                            Ethanol        0.7                                                            MPG            2.7 (Mono Propylene Glycol)                                    Glycerol       0.5                                                            Sodium sulfate                                                                               0.4                                                            Sodium carbonate                                                                             2.7                                                            Sodium citrate                                                                               4.4                                                            Citric acid    1.5                                                            Water         60.8                                                            ______________________________________                                    

Enzyme dosage: 1% w/w Saviase (14 KNPU/g)+1% w/w Lipolase (100 KLU/g)

Enzyme Stabilizer Dosage: 5 mmole/kg (for boric acid 160 mmole/kg)Storage: 0, 3, 7 and 14 days at 30° C.

The results of the inhibition effectiveness IF_(I) of the boronic andborinic acid derivative enzyme stabilizers tested are listed below.

                  TABLE 2                                                         ______________________________________                                        Different Boronic and Borinic Derivatives Enzyme Stabilizers                  and the Corresponding IF.sub.I. Boric Acid is Included for Comparison.        Inhibitor        Improvement factor IF.sub.I                                  Boric acid       1                                                            Thiophene-2-boronic acid                                                                       72                                                           Thiophene-3-boronic acid                                                                       63                                                           4-Methylthiophene-2-                                                                           4                                                            boronic acid                                                                  5-Ethylthiophene-2-                                                                            66                                                           boronic acid                                                                  5-Bromothiophene-2-                                                                            124                                                          boronic acid                                                                  5-Chlorothiophene-2-                                                                           123                                                          boronic acid                                                                  Dibenzothiophene-1-                                                                            12                                                           boronic acid                                                                  Dibenzofuran-1-  9                                                            boronic acid                                                                  5-Methylthiophene-2-                                                                           12                                                           boronic acid                                                                  5-Methoxythiophene-2-                                                                          14                                                           boronic acid                                                                  Furan-2-boronic acid                                                                           13                                                           Dimethylthiophene-3-                                                                           7                                                            boronic acid                                                                  Furan-3-boronic acid                                                                           32                                                           3-Methoxythiophene-2-                                                                          8                                                            boronic acid                                                                  Benzofuran-2-boronic acid                                                                      190                                                          Thionaphthrene-1-                                                                              20                                                           boronic acid                                                                  Dibenzofuran-4-boronic                                                                         19                                                           acid                                                                          ______________________________________                                    

Comparing the results of Table 1 with the results of Table 2 it seemsthat the effect of a boronic or a borinic acid stabilizer in a detergentcan be predicted from the results obtained in buffer systems and viceversa.

We claim:
 1. A liquid composition comprising an enzyme and a boronicacid derivative enzyme stabilizer of the following formula: ##STR3##where R₁ is: (a) a substituted fused aromatic ring structure containing14 or 18 carbon atoms in the ring;(b) a substituted monocyclicheterocyclic ring structure containing up to 17 carbon atoms in thering; (c) a substituted fused aromatic heterocyclic ring structurecontaining up to 17 carbon atoms in the ring; (d) a substitutedmonocyclic quinonone ring structure containing up to 18 carbon atoms inthe ring; (e) a substituted fused quinonoid ring structure containing upto 18 carbon atoms in the ring; wherein substitutions are selected fromthe group consisting of hydrogen, C₁ -C₆ alkyl, aryl, hydroxy, halogen,amine, alkylated amine, nitro, thiol, aldehyde, acid, acid salt, ester,sulfonate, and phosphonate.
 2. The liquid composition according to claim1, wherein the enzyme stabilizer is thiophene-2-boronic acid orthiophene-3-boronic acid.
 3. The liquid composition according to claim2, wherein the thiophene group is substituted with bromine or chlorine.4. The liquid composition according to claim 1, wherein the enzymestabilizer is 5-bromo-thiophene-2-boronic acid or5-chloro-thiophene-2-boronic acid.
 5. The liquid composition accordingto claim 2, wherein the thiophene group is substituted with methyl orethyl.
 6. The liquid composition according to claim 5, wherein theenzyme stabilizer is 4-methyl-thiophene-2-boronic acid,5-methyl-thiophene-2-boronic acid or 5-ethyl-thiophene-2-boronic acid.7. The liquid composition according to claim 1, wherein the enzymestabilizer is dibenzothiophene-1-boronic acid.